Field
The invention relates to a modular system and a method for the continuous, microbe-reduced production and/or processing of a product from a heterogeneous cell culture-fluid mixture.
Description of Related Art
In biotechnological production, proteins are usually purified in batches. This means that the individual production cycles are handled discontinuously in a batchwise manner, with the entire product being removed after completion of a production cycle. To produce again, it is then necessary to start a separate new product cycle/ batch.
In recent years, it has been increasingly demonstrated that a continuous procedure can also be performed in biotechnological production, where the process runs without interruptions, in contrast to a batch process.
The highly regulated pharmaceutical production requires great effort in terms of time, technology and personnel to provide cleaned and sterilized bioreactors and to ensure a sterile product. To reliably avoid cross-contamination in the event of a product changeover in a multipurpose system or between two product batches, what is required apart from cleaning is a very complex cleaning validation, which, if applicable, must be repeated in the event of a process adaptation.
This applies both to upstream processing (USP), i.e. the production of biological products in fermenters, and to downstream processing (DSP), i.e. the purification of the fermentation products.
Especially in the case of fermentation, a sterile environment is essential for a successful culture.
To sterilize batch fermenters or fed-batch fermenters, the SIP technique (SIP =sterilization-in-place) is generally used.
The downtime of reactors resulting from the necessary cleaning and sterilization procedures can take up a significant share of reactor availability, especially in the case of short usage periods and frequent product changes. This affects, for example, the process steps of media preparation and fermentation in USP of biotechnological production, and solubilization, freezing, thawing, pH adjustment, production separation, e.g. via chromatography, precipitation or crystallization, adjusting buffers and virus inactivation in DSP.
In the downstream process, the regulatory requirements are a microbe-reduced process management. Therefore, there is no need for a sterile process in the case of batch operation.
However, in a continuous process, the purification of the protein is performed over a relatively long period of time if possible without cleaning steps. This preferably occurs without sterilization steps during the purification. This is the case even though the risk of microbial contamination is many times higher than in the case of a simple batch operation.
WO2012/078677 describes a process and a system for the continuous processing of biopharmaceutical products by means of chromatography and the integration thereof in a production system, more particularly in a disposable system. Although WO2012/078677 provides approaches for the continuous production of biopharmaceutical and biological products, the disclosed solution is not adequate in practice. WO2012/078677 also does not disclose the use of a sterilized chromatography column.
US 2014/0255994 A1 discloses an integrated continuous process for producing therapeutic proteins. However, US 2014/0255994 A1 does not disclose the feature that sterilized chromatography columns could be used in such a process.
EP 2 182 990 A1 discloses a process for sterilizing chromatography columns by using hot water vapour.
First of all, some terms will be defined in more detail.
In the context of this invention, a continuous process means any process for carrying out at least two process steps in series, the output stream of an upstream step being conveyed to a downstream step in said process. The downstream step starts the processing of the product stream before the upstream step has been completed. Typically, in a continuous process, part of the product stream is always being conveyed in the production system and is referred to as a “continuous product stream”. Accordingly, a continuous conveyance or transfer of a product stream from an upstream unit to a downstream unit means that the downstream unit is already operating before the upstream unit is put out of operation, i.e. that two successively connected units simultaneously process the product stream flowing through them.
In the context of the invention, the term “microbe-reduced” means a state of reduced microbial count, i.e. a microorganism count per unit area or unit volume of virtually zero, which is achievable by a suitable microbe-reduction method, it being possible to select said microbe-reduction method from gamma irradiation, beta irradiation, autoclaving, ethylene oxide (ETO) treatment and “steam-in-place” (SIP) treatment.
In the context of the invention, the term “disposable article” means that the articles in question that come into contact with product, more particularly apparatuses, tanks, filters and connecting elements, are suitable for one-time use with subsequent disposal, it being possible for said tanks to be made both from plastic and from metal. In the context of the invention, the term also encompasses reusable articles, for instance made of which are used only once in the process according to the invention and are then no longer used in the process. In the context of the invention, said reusable articles, made of steel for example, are then also referred to as “objects used as disposable articles”. Such employed disposable articles can also be referred to as “disposable” or “single-use” articles (“SU technology”), respectively, in the process according to the invention. These yet further improve the microbe-reduced state of the process according to the invention and of the modular system.
In the context of the invention, the term “product stream” means the particle-free fluid from a heterogeneous cell culture/fluid mixture containing the product, and the result of each of the other process steps of the process according to the invention, i.e. the product stream after filtration, after chromatography, after virus depletion, after ultrafiltration, after diafiltration, or after further steps of the process according to the invention, it then being possible for said product streams to have different concentrations and degrees of purity.
In the context of the invention, the term “virus depletion” means a reduction in the concentration of active viruses per unit volume of the fluid to be treated, right up to complete inactivation and/or removal of the viruses present in the fluid to be treated.
In the context of the invention, the term “microbicide” means a substance which can slow or completely inhibit the growth of microorganisms, it being possible for said microbicide to be used in the form of a microbicide-containing buffer, especially during an ultrafiltration in the context of the process according to the invention.
In the context of the invention, the term “bubble trap” means a device for collecting gas bubbles while the fluid in question is degassed at the same time, with the fluid in question being degassed when this is taking place.
In the context of the invention, the term “modular” means that the individual steps of the process according to the invention can be carried out in separate modules that are connected to one another, the modules being preconfigured and microbe-reduced and it being possible to connect them to one another in a closed manner and in different combinations.
In the context of the invention, the term “modular system” means a series of modules (“units”) in which a fluid (“product stream”) can be conveyed and which are connected to one another for carrying out at least two downstream and/or upstream steps. According to the invention, the units are suitable for continuously carrying out a step and can be operated with a continuous fluid stream (“product stream”). In this connection, the individual modules of the “modular system” can be connected to one another in any combination. Examples of modules in the context of the invention are the filtration module 2, the chromatography module 3, the ultrafiltration module 6, the diafiltration module 7 and the dialysis module 8.
In the context of the invention, the term “closed” means the mode of operation of the process according to the invention and of the modular system according to the invention, which are operated such that the product produced and/or processed by said process and said modular system is not exposed to the room environment. Materials, objects, buffers and the like can be added from the outside to the closed process according to the invention and the corresponding closed modular system according to the invention, however, this addition takes place in such a way that an exposure of the produced and/or processed product to the room environment is avoided.
The processes known from the prior art have a range of disadvantages, which will be dealt with below.
Known processes for producing biopharmaceutical and biological products typically comprise the following production steps, which are connected to one another:                1. perfusion culture        2. cell retention system,as an alternative to steps 1 and 2, also a feed-batch culture may be employed,        3. cell removal        4. buffer or media exchange, preferably with concentration        5. bioburden reduction, preferably by sterile filtration        6. capture chromatography.        
Typically, further steps are carried out for further purification of the product stream, more particularly:                7. virus inactivation        8. neutralization, and        9. optionally a further depth filtration, bioburden reduction (sterile filtration).        
In view of the high quality standards in the production of biopharmaceuticals, the following steps are typically additionally carried out:                10. chromatographic intermediate and high-quality purification        11. bioburden reduction, for example sterile filtration        12. virus filtration        13. buffer exchange and preferably concentration, and        14. sterile filtration.        
In the above-described production, cells in a fermenter containing nutrient solution produce a biological product, for instance a protein, for example a therapeutic protein. The nutrient solution is also an ideal growth medium for microorganisms, such as bacteria and spores. As this growth of such microorganisms is not desired a problem arises from these circumstances. Said undesired growth of microorganisms especially becomes a problem in the case of relatively long run times because the nutrient solution becomes increasingly contaminated as the run time of the process increases, right up to an exponential growth of microorganisms and thus a total loss of the batch of the biological product that is produced.
To cope with the demand for a rapid and flexible reloading of the production system while maintaining maximum cleanliness and sterility, concepts for a continuous production, preferably using disposable technology, are attracting a constantly growing interest in the market.
For relatively long run times of such a process, ranging from two or more hours over days to weeks, customary sanitization measures are, however, insufficient, for example the customary “clean-in-place” (CIP) measures, such as sanitization by means of 1 M NaOH for example. In the case of run times above two or more hours, such customary processes and systems therefore have the disadvantage that they are highly susceptible to possible contamination and/or possible microbial growth.
Therefore, there is a need for a process for the continuous purification of a product from a heterogeneous cell culture-fluid mixture, which due to its microbe-reduced state allows a continuous mode of operation for several weeks.